Methotrexate (MTX) is an antifolate that is used essentially in all treatment protocols for childhood acute lymphoblastic leukemia (ALL). After its entry into cells, MTX is rapidly converted to γ-glutamyl polyglutamates through the action of folypolyglutamate synthetase (FPGS). Long chain polyglutamates (MTXPG4-7) are more avid inhibitors of folate-dependent enzymes and are also retained longer within cells, thereby increasing and prolonging MTX's antifolate effects. Higher accumulation of MTXPG has been associated with increased cytotoxicity and treatment response in childhood ALL.
Significant lineage and ploidy differences have been observed in MTX-PG accumulation in ALL cells, with T-lineage ALL having the lowest MTX-PG accumulation and hyperdiploid (>50 chromosomes) and B-lineage ALL having the highest MTX-PG accumulation. The underlying mechanisms for these differences include lower FPGS activity in T-ALL. However, following uniform treatment with HDMTX, there remain substantial inter-individual differences in MTX-PG accumulation within each of the three lineage and ploidy subtypes of ALL, for reasons that have not been fully elucidated.
One potential cause of inter-individual differences in MTX-PG accumulation is heterogeneity in γ-Glutamyl hydrolase (GGH, also known as folypolyglutamate hydrolase, FPGH, EC 3.4.19.9), a lysosomal peptidase that catalyzes the removal of γ-linked polyglutamates, converting long-chain MTX-PG into shorter-chain MTX-PG and ultimately to MTX. This allows MTX to be effluxed from cells and thereby reduces the overall effectiveness of MTX. The human GGH gene spans 24 kb on chromosome 8 (q12.23-13.1) and comprises nine exons (Yin, D. et al., “Structural organization of the human gamma-glutamyl hydrolase gene” Gene 238: 463-470 (1999)). The crystal structure of human GGH has been determined and a model for substrate recognition and hydrolysis has been proposed (Li, H. et al., “Three-dimensional structure of human gamma-glutamyl hydrolase. A class I glatamine amidotransferase adapted for a complex substate” J Biol Chem 277: 24522-24529 (2002); Chave, K. J, et al., “Molecular modeling and site-directed mutagenesis define the catalytic motif in human gamma-glutamyl hydrolase” J Biol Chem 275: 40365-40370 (2000)). Cellular GGH is predominantly lysosomal, with an acidic pH optimum, functioning as either an endopeptidase or exopeptidase, exhibiting species differences in these functions. Human GGH has a higher affinity for the longer chain MTX polyglutamates, cleaving multiple glutamyl residues, having its highest activity at the outermost or two outermost residues in the polyglutamate chain (Panetta, J. C., et al., id).
Polymorphisms within the GGH gene have been reported (Chave, K. J. et al., “Identification of single nucleotide polymorphisms in the human gamma-glutamyl hydrolase gene and characterization of promoter polymorphisms”. Gene 319: 167-175 (2003)). These polymorphisms which occurred in the promoter region of the GGH gene were reported as potentially affecting expression of the GGH protein, while a polymorphism occurring in the coding region which caused a codon change (452 C>T; T127I) was reported as not changing GGH activity (Chave, K. J. et al., 2003, id). This report indicates that GGH promoter polymorphisms may play a role in inter-individual differences in MTX-PG accumulation, but that the coding region polymorphism does not since it did not change GGH activity.